Because serotonergic function has been implicated in the pathophysiology of a number of diseases of the nervous system, efforts to image this system in vivo have received considerable recent attention. Promising preliminary results with the tracer 5-iodo-6-nitroquipazine (INQUIP) have prompted us to perform further studies designed to validate the use of the tracer as an in vivo ligand for the serotonin transporter. We studied 6 adult Macaca mulatta in 8 experiments which involved SPECT imaging at 17 to 24 hours post tracer injection, including 3 experiments with coinjection of the 123I and 125I radiolabeled tracer for direct comparison of autoradiography and SPECT, and 3 experiments in which animals were lesioned with the serotonergic neurotoxin (1)3,4-methyl-enedioxymethamphetamine (MDMA). In addition, we evaluated the metabolism of the tracer in the brain and periphery. SPECT images obtained at 17 and 24 hours reflected the known pattern of distribution of serotonin transporters and also showed close correspondence to the autoradiograms. Ratios of binding in the brainstem to binding in the cerebellum were close to 3 at 17 hours. Autoradiograms from an MDMA treated animal showed up to 95% reductions of binding, while the SPECT data showed smaller reductions. Virtually all of the tracer in the brainstem was in the form of unmetabolized parent compound, but plasma showed rapid peripheral metabolism of the tracer. These results demonstrate that INQUIP SPECT images are sensitive measures of in vivo binding to the serotonin transporter, and support the further development of the tracer as a method for the in vivo study of serotonergic neurons in humans.